Effect of SGLT2 inhibition on salt-induced hypertension in female Dahl SS rats

Sodium-glucose co-transporters (SGLTs) in the kidneys play a pivotal role in glucose reabsorption. Several clinical and population-based studies revealed the beneficial effects of SGLT2 inhibition on hypertension. Recent work from our lab provided significant new insight into the role of SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension, Dahl salt-sensitive (SS) rats. Dapagliflozin (Dapa) blunted the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the Renin–Angiotensin–Aldosterone System. However, our initial study used male SS rats only, and the effect of SGLT2 inhibitors on hypertension in females has not been studied. Therefore, the goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function in female Dahl SS rats. The result showed that administration of Dapa for 3 weeks prevented the progression of salt-induced hypertension in female rats, similar to its effects in male SS rats. Diuresis and glucose excretion were significantly increased in Dapa-treated rats. SGLT2 inhibition also significantly attenuated kidney but not heart fibrosis. Despite significant effects on blood pressure, Dapa treatment caused minor changes to electrolyte balance and no effects on kidney and heart weights were observed. Our data suggest that SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension independent of sex.


Dapa treatment mitigates the progression and intensity of salt-induced hypertension in female Dahl SS rats
To test an effect of SGLT2i on blood pressure during the progression of salt-induced hypertension, we fed Dahl SS rats with 4.0% NaCl high-salt (HS) diet for three weeks (see protocol in Fig. 1A).At baseline, while the animals were fed a 0.4% NaCl normal salt (NS) diet, the mean arterial blood pressure (MAP) between the vehicle and Dapa groups was not statistically different from each other (129 ± 1 versus 124 ± 2 mmHg, respectively).Exposure to a HS diet for three weeks caused a notable rise in blood pressure, reaching 160 ± 3 mmHg for the group treated with a vehicle but Dapa treatment prevented the increase to levels seen in vehicle treatment (139 ± 7 mmHg) (P < 0.05).Throughout the final week of the study (D13-D21), the Dapa-treated rats displayed a markedly reduced blood pressure, maintaining ~ 20 mmHg lower by the end of the experiment (Fig. 1B and C; D21, P < 0.001).A similar pattern was seen for systolic (SBP) and diastolic blood pressure (DBP) measurements when comparing Dapa-treated to vehicle-treated rats (Fig. 1D and E); however, the statistically only SBP was different at the end of the experiment.No differences in circadian variations of MAP (Fig. 1F) and heart rate (Fig. 1G) were observed between groups.
Kidney and heart weights were analyzed to determine if Dapa had an effect on organ weights.No significant difference was found in body weight between the control and experimental groups (Fig. 2A).Also, the ratio of kidney weight to body weight (2Kidney/BW) was not different in the Dapa-treated group compared to the vehicle-treated rats at the end of the experiment (Fig. 2B).Dapa-treated rats showed a significantly lower heart weight; however, this difference was lost when comparing heart weight to body weight ratios (Fig. 2C).
Consistent with the pharmacodynamics of SGLT the administration of Dapa increased glucose excretion.This was associated with ~ two-fold greater diuretic effect compared to vehicle on D14 (P < 0.05, Fig. 2D).Moreover, urinary glucose concentration normalized to urinary creatinine and absolute glucose excretion were significantly elevated in Dapa-treated rats to vehicle-treated (182 ± 18 versus 0.3 ± 0.03 mg/dL and 101 ± 17 versus 0.18 ± 0.03 mg/day on D21, respectively, P < 0.001; Fig. 2E).

Dapa treatment does not affect electrolyte homeostasis in female rats
Dapa treatment did not alter the urinary electrolytes to creatinine ratios on D7, D14 compared to a vehicle (Fig. 3A-D).However, Na + , Cl − and Ca 2+ excretion were significantly higher in Dapa-treated rats at the end of the experimental period (Fig. 3A, B and D, respectively).No significant differences in urinary K + to creatinine ratios were observed between the groups (Fig. 3C).An estimation of fractional excretion showed no differences between groups (Na + , K + , Cl − , Ca 2+) (Fig. 3E).By the end of the experimental protocol, no significant changes were observed between groups in plasma electrolytes and creatinine and non-fasting blood glucose (Table 1).Fasting blood glucose levels were not measured to avoid changes in blood pressure due to caloric restriction.

Dapa treatment does not affect creatinine clearance but attenuates kidney fibrosis in female rats
Subsequently we examined whether the beneficial effect of Dapa treatment on MAP resulted in concurrent effects on kidney function and damage.Creatinine clearance did not show significant differences between Dapa and vehicle groups (Fig. 4A).Consistent with this, urinary albumin excretion (Fig. 4B), as well as urinary albuminto-creatinine ratio (Fig. 4C) and plasma creatinine (Table 1), did not show significant differences between groups.Subsequently, we evaluated the influence of SGLT2 inhibition on structural kidney damage.Examination of Masson's Trichrome stained kidney sections showed that protein casts did not vary significantly between the groups.However, semiquantitative analysis showed lower cortical fibrosis in the treated group (Fig. 5A-C).

Dapa treatment does not affect the amount of collagen in the hearts of female rats
To evaluate the degree of myocardial collagen, we used Picrosirius Red staining imaged with polarized light to highlight collagen and selected multiple frames from different cardiac regions.This analysis of stained sections showed that there was no significant difference in the left, right ventricular or septum cardiac connective tissue amount between Dapa-and vehicle-treated rats (Fig. 5D, E).

Discussion
In the current study, we determined the effects of the SGLT2i Dapa on the development of hypertension and severity of target-organ damage in female Dahl SS rats.This study adds valuable information and complements our previous studies, which predominantly focused on male rats 16 .It is known that animal models of salt-sensitive hypertension exhibit lower blood pressure in females than in males [5][6][7] .Here we show that Dapa administration to female rats did not differ significantly from males in the magnitude of blood pressure reduction, which we reported previously 16 .The rationale for completing these experiments is due to known sex differences in renal transporter expression 17 .Differences in SGLT2 expression were observed between female and male rodents.In C57BL/6 mice, SGLT2 protein expression is lower in females compared to males; however, Sglt2 mRNA expression showed the opposite 18 .In contrast, in Wistar rats, SGLT2 protein expression was higher in females compared to males 18,19 and Sglt2 mRNA expression in the renal cortex was similar 18 .In human kidneys no differences in SGLT2 expression were reported 20 .
While SGLT2 is not a major contributor to sodium transport in the kidneys, its inhibition was associated with several pleiotropic effects on electrolytes in distal nephron segments, particularly in the distal convoluted tubule and the collecting duct.Female rats have more rapid diuretic and natriuretic responses to saline load, possibly due to differences in transporter expression in proximal and distal nephrons 17  cotransporter 2, aquaporin-1, and claudin-2 in the proximal tubule together with lower Na + and HCO 3 − reabsorption and therefore increased flow from out of the proximal tubule.In distal segments of the nephron, female rats have a higher abundance of total and phosphorylated Na + -Cl − cotransporter (NCC), claudin-7, and cleaved forms of α-and γ-ENaC subunits 17 .At variance to our findings in male Dahl SS rats 16 , the diuretic effect of Dapa was less pronounced in females.If this relates to differences in SGLT2 expression or differences in the compensatory responses in further downstream segments remains to be determined.
Interestingly, one of the major differences we observed between males and females that were treated with Dapa was the kidney-to-body weight ratios.In the male treatment group, there was a significant increase in the kidney-to-body weight ratios 16 .In contrast, the Dapa-treated females did not exhibit this change.It has been proposed that SGLT2i treatment can increase kidney weights by increasing the cellular area of the S3 proximal tubule and collecting duct nephron segments 21 .This study, however, only investigated this change in male mice, and not females.It would be of additional physiological significance to look into this sex difference more closely in the future, as it may be of clinical interest.
Our results demonstrate that Dapa administration reduced the magnitude of blood pressure increase in female salt-sensitive rats without renal electrolyte handling or kidney hypertrophy.Retrospectively comparing current results to the previous study in male 16 , female Dahl SS rats showed the same amplitude in blood pressure changes under Dapa treatment (~ 20 mmHg) despite the similar MAP pressure (Table 2).This data suggests that dapagliflozin blunts salt-sensitive hypertension both in male and female rats.Studies investigating the effect of SGLT2i on blood pressure and renal function in female subjects are limited but have provided some insight into their effects in females.In one study, empagliflozin treatment of hyperandrogenic female rats (polycystic ovary syndrome model) showed only a small decrease in blood pressure, and no effect on albuminuria was observed 22 .In female db/db mice, empagliflozin improved diastolic function, reduce cardiomyocyte cross-sectional area, myocardial fibrosis, and pro-fibrotic SGK1/ENaC protein expression levels but not blood pressure or dipping status 23 .It is worth noting that SGLT2i also have potential implications for the treatment of preeclampsia.One study employing AT1-AA-induced preeclampsia in mice showed that empagliflozin might have a protective effect on preeclampsia by ameliorating systolic blood pressure, proteinuria, podocyte injury and oxidative stress 24 .
The renoprotective properties of SGLT2i are well-established.Numerous preclinical and clinical studies have highlighted the connection between SGLT2i treatment and a decrease in various markers of inflammation and kidney injury 25,26 , as well as preservation of kidney function decline in CKD [27][28][29] .Animal-based research has also indicated the renoprotective effects of gliflozins [30][31][32][33] .In Angiotensin II-dependent hypertension, empagliflozin www.nature.com/scientificreports/administration prevented the development of renal fibrosis without affecting blood pressure 34 .Empagliflozin also suppressed kidney fibrosis, renal growth and inflammation in diabetic mice 33,35 .The beneficial role of SGLT2i was also shown to reduce the risk of heart failure progression or mortality in patients regardless of the presence of diabetes mellitus 36 .Our experiments showed a noticeable decrease in cortical fibrosis but not protein cast development after Dapa treatment in female Dahl SS rats.However, no specific improvements to cardiac fibrosis or heart rate were observed.In summary, our findings demonstrate that administration of Dapa prevents salt-induced hypertension in female rats and reduces renal cortical tissue fibrosis but does not affect electrolyte excretion or cardiac damage.Our findings thus highlight the importance of including sex as a biological variable in pharmacological research.This factor is particularly important in studies involving diseases that affect men and women differently.

Experimental protocol and animals
Animal experiments and procedures adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and reported in accordance with ARRIVE guidelines.The protocols were reviewed and approved by the University of South Florida (USF) Institutional Animal Care and Use Committee.Female Dahl salt-sensitive rats (SS; SS/JrHsdMcwi; RRID: RGD_61499) were fed normal salt (NS) diet (0.4% NaCl; #113755; Diets Inc., New Brunswick, NJ).For the high salt challenge, rats were placed on 4% NaCl high salt (HS) diet (D113756; Diets Inc.).Rats were maintained on an HS diet for 3 weeks and treated with Dapa or vehicle.Dapa (Cat.464620010; ThermoFisher Scientific, Waltham, MA) was added to the drinking water at ~ 2 mg/kg/ day (the stock solution was prepared in ethanol in a concentration of 20 mg/ml).The concentration of the drug in water was adjusted with the increased water consumption to keep a constant dose during the experiment.Water and food were provided ad libitum.The treatment was initiated on the first day of the HS challenge, as shown in previously published protocols 16 .

Surgical procedures
At the age of 8-8.5 weeks, rats were anesthetized on a temperature-controlled platform via inhalation of 2.5% isoflurane in 0.5 L/min (O 2 /N 2 : 30%/70%).A blood pressure transmitter (HD-S10; Data Sciences International, New Brighton, MN) was implanted subcutaneously.The catheter tip was secured in the abdominal aorta via the femoral artery 37,38 .Rats were allowed to recover for 4-5 days, and blood pressure and heart rate were recorded using DSI software.At the end of the experiment timeline, rats were anesthetized by 5% isoflurane, and kidneys were flushed with phosphate-buffered saline via aortic catheterization [39][40][41] .The left kidney was snap-frozen, and the right kidney was placed in 10% formalin for histological studies.

Histological analysis
Rat kidneys and hearts were formalin-fixed, paraffin-embedded, sectioned, and mounted on slides as previously described 41,42 .Slides were processed with Masson's trichrome stain.Protein cast and fibrosis analysis were assessed in whole kidney slides scanned at × 10 magnification using color deconvolution and thresholding in QuPath-0.4.2 software 43 .Fibrosis was quantified for the cortex area, and protein casts for the outer medulla area.
Collagen fibers in the heart tissue sections were visualized using Picrosirius Red staining under polarized light microscopy.For semi-quantification of collagen fibers, each region-of-interest was drawn on the middle third of the free walls of both ventricles (right (RV) and left ventricle (LV) and septum (S) based on the inner junctional points (red asterisk) (Fig. 5E).

Statistics
Statistical analysis was performed following recent guidelines 44 .The data are displayed as the mean ± the standard error of the mean (SEM).In the box plot illustrations, the box signifies ± SEM.The data underwent tests for normality using the Shapiro-Wilk method and for equal variance via Levene's homogeneity test.Statistical evaluations were carried out using ANOVA or t-tests (GraphPad Prism 9.0), considering a P-value of less than 0.05 to indicate significance.Moreover, if the results of an ANOVA test were significant, a post hoc analysis was conducted using Holm-Sidak's multiple-comparison method.

Figure 1 .
Figure 1.The effect of dapagliflozin treatment on magnitude of blood pressure in female Dahl SS rats.(A) Schematic representation of the experimental protocol.(B) The mean arterial pressure (MAP) in SS rats after diet change from normal salt (NS, 0.4% NaCl; day 0) to a high salt (HS, 4% NaCl) diet and chronic treatment with the SGLT2 inhibitor (dapagliflozin) or vehicle (C) Changes in MAP in weeks 1, 2, and 3 of Dapa treatment on the HS diet compared to baseline MAP on the NS diet.Systolic (SBP) (D) and diastolic (DBP) (E) blood pressure in SS rats after diet change from normal salt (NS, 0.4% NaCl; day 0) to a high salt (HS, 4% NaCl) diet and chronic treatment with the SGLT2 inhibitor (dapagliflozin) or vehicle.(F) Diurnal blood pressure and (G) heart rate recordings on HS diet over the last three days, each point is a 2-h average.Shaded regions in (F) and (G) indicate the active period for animals (lights were off in the animal facility).For all figures n = 6-7, two-way repeated-measures ANOVA was used, *P < 0.05.** P < 0.01, *** P < 0.001 are considered statistically significant.

Table 1 .
Blood parameters in female SS rats after 21 days of chronic experiment with dapagliflozin treatment on HS.
Urine was collected for 24 h in metabolic cages (no.37000M071, Tecniplast, West Chester, PA) at baseline and every 7 days of the HS protocol.Prior to euthanasia, blood samples were collected by aortic catheterization in anesthetized animals.Glucose, creatinine, and electrolytes (Na + , K + , Ca 2+ , Cl − ) in plasma and urine were measured with a blood gas analyzer (ABL system 800 Flex, Radiometer, Copenhagen, Denmark).Fractional excretion (FE) of electrolytes was calculated by the formula FEX (%) = [Urine electrolyte concentration (mmol/L, or mg/ dL) × serum creatinine (mg/dL) divided by serum electrolyte concentration (mmol/L, or mg/dL) × urine creatinine (mg/dL)] × 100%, where the variable 'X' represents the specific electrolyte under consideration.Urine albumin was determined by a fluorescent assay (Albumin Blue 580 dye, Molecular Probes, Eugene, OR) using a fluorescent plate reader (Synergy Neo 2, Bio-Tek, Winooski, VT).

Table 2 .
16mparing of the effect of dapagliflozin on blood pressure in male and female SS rats.*Experiments were performed at different time, retrospective data for male Dahl SS16was used.